The long-term goal of this research is to gain an integrated view of how S. meliloti establishes the chronic intracellular infection that underlies its symbiosis and to use these findings to gain insights into the molecular strategies used by certain chronic intracellular mammalian pathogens. The proposal includes experiments analyzing a gene (YbeY/Smc01113), originally discovered because of its importance for symbiosis, that is present in every bacterium and encodes a previously undiscovered RNase whose function is required for the maturation of all three ribosomal rRNAs - 16S, 23S, and 5S. The human homolog of YbeY, C21ORF57, encoded in the Down Syndrome Critical Region of chromosome 21, is also an RNase that is critically required for mitochondrial function in human cells. We will to continue to investigate the physiological roles of YbeY, including characterizing its relationship to other RNases and investigating its interaction with certain ribosome maturation factors. A high-throughput screen has been validated and will be used to screen for small molecule YbeY inhibitors that could serve as lead compounds for the development of a new antibiotic. We will continue to characterize the biochemical properties of YbeY, its interactions with partner proteins, and determine the structure of YbeY in a complex with an RNA substrate. We will continue to use cell lines and transgenic mice to investigate the roles of the human YbeY homolog C21ORF57 and a possible connection to Down syndrome. We will continue our efforts to determine the role of bacterial cell cycle control in bacteroid differentiation of S. meliloti during symbiosis, including analyzing the importance of proteolytic control of the key cell cycle regulator CtrA. We will continue to investigate other functions important for S. meliloti symbiosis and their possible roles in Brucella pathogenesis, including determining whether certain genes essential for S. meliloti symbiosis are also necessary for Brucella pathogenesis and analyzing the importance of small RNA regulation in S. meliloti symbiosis. There is a critical need for new antibiotics since only three new classes of antibiotics have been discovered since 1962 and the emergence of antibiotic-resistant and "superbugs" threatens our ability to treat infectious diseases. A drug that inhibits YbeY could be a new class of broad-spectrum antibiotic. If C21ORF57 proves to contribute to the pathology of trisomy 21, an inhibitor of C21ORF57 could be the first drug to treat Down syndrome. Work on S. meliloti symbiosis is helping to identify Brucella genes necessary for pathogenesis that could be new drug targets. This is important because Brucella infections are difficult to treat and Brucella is regarded as a bioterrorism threat. Brucella mutants whose virulence is appropriately attenuated could be vaccine candidates as there is currently no vaccine against Brucella. The project has offered novel insights into the biosynthesis of B12, a critically important vitamin for human health, and will continue to explore its role in biology. Finally, our work on the S. meliloti-Medicago symbiosis serves as a model for many symbioses found in Nature. PUBLIC HEALTH RELEVANCE: A drug that inhibits YbeY could be a new class of broad-spectrum antibiotic and, if C21ORF57 proves to contribute to the pathology of trisomy 21, an inhibitor of C21ORF57 could be the first drug to treat Down syndrome. Work on S. meliloti symbiosis is helping to identify Brucella genes necessary for pathogenesis that could be new drug targets. The project has offered novel insights into the biosynthesis of B12, a critically important vitamin for human health, and will continue to explore the role of vitamin B12 in biology.